Ferredoxin-NADP + reductases (FNRs) are ubiquitous flavoenzymes involved in redox metabolisms. FNRs catalyze the reversible electron transfer between NADP(H) and ferredoxin or flavodoxin. They are classified as plant-and mitochondrial-type FNR. Plant-type FNRs are divided into plastidic and bacterial classes. The plastidic FNRs show turnover numbers between 20 and 100 times higher than bacterial enzymes and these differences have been related to their physiological functions. We demonstrated that purified Escherichia coli FPR (EcFPR) contains tightly bound NADP + , which does not occur in plastidic type FNRs. The three-dimensional structure of EcFPR evidenced that NADP + interacts with three arginines (R144, R174, and R184) which could generate a very high affinity and structured site. These arginines are conserved in other bacterial FNRs but not in the plastidic enzymes. We have cross-substituted EcFPR arginines with residues present in analogous positions in the Pisum sativum FNR (PsFNR) and replaced these amino acids by arginines in PsFNR. We analyzed all proteins by structural, kinetic, and stability studies. We found that EcFPR mutants do not contain bound NADP + and showed increased K m for this nucleotide. The EcFPR activity was inhibited by NADP + but this behavior disappeared as arginines were removed. A NADP + analog of the nicotinamide portion produced an activating effect on EcFPR and promoted the NADP + release. Our results give evidence for a new model of NADP + binding and catalysis in bacterial FNRs.We propose that this tight NADP + binding constitutes an essential catalytic and regulatory mechanism of bacterial FNRs involved in redox homeostasis. K E Y W O R D S catalytic mechanism, catalytic site, Escherichia coli, ferredoxin-NADP + reductase, NADP + binding 1 | INTRODUCTION Ferredoxin-NADP + reductases (FNRs) constitute a family of monomeric hydrophilic proteins that contain noncovalently bound FAD as a prosthetic group. These enzymes are widely distributed among organisms and are